Abstract

Phallusia mammillata has recently emerged as a new ascidian model. Its unique characteristics, including the optical transparency of eggs and embryos and efficient translation of exogenously introduced mRNA in eggs, make the Phallusia system suitable for fluorescent protein (FP)-based imaging approaches. In addition, genomic and transcriptomic resources are readily available for this ascidian species, facilitating functional gene studies. Microinjection is probably the most versatile technique for introducing exogenous molecules such as plasmids, mRNAs, and proteins into ascidian eggs/embryos. However, it is not practiced widely within the community; presumably, because the system is rather laborious to set up and it requires practice. Here, we describe in as much detail as possible two microinjection methods that we use daily in the laboratory: one based on an inverted microscope and the other on a stereomicroscope. Along the stepwise description of system setup and injection procedure, we provide practical tips in the hope that this chapter might be a useful guide for introducing or improving a microinjection setup.

Supplementary material

Movie 3.1Unfertilized Phallusia egg during microinjection with mRNA. Images created using an inverted microscope (Olympus IX70) and a ×10 objective lens. To show the difference between a dead Phallusia egg and a live Phallusia egg, a live egg is moved next to a dead egg (which appears dark in the movie) using the injection needle. The needle is inserted into the live egg a little more than half way; then, using the high-pressure injection system, a 100-ms air pulse is activated, which forces some mRNA into the egg, causing a small displacement of the cytoplasm at the tip of the needle (a transient clear zone appears, arrow at 6 s). Using the stage control, the needle is then removed rapidly from the egg at the end of the movie. The dark zone to the right is the VALAB of the wedge. Scale bar = 50 μm (AVI 49468 kb)